Chang Baolei, Su Yanyu, Li Tingting, Zheng Yanxia, Yang Ruirui, Lu Heng, Wang Hao, Ding Yusong
College of Public Health, Xinjiang Medical University, Urumqi 830011, Xinjiang, China;Department of Preventive Medicine, School of Medicine, Shihezi University, Shihezi 832003, Xinjiang, China;Key Laboratory for Prevention and Control of Emerging Infectious Diseases and Public Health Security, the Xinjiang Production and Construction Corps, Shihezi 832003, Xinjiang, China.
Department of Preventive Medicine, School of Medicine, Shihezi University, Shihezi 832003, Xinjiang, China;Key Laboratory for Prevention and Control of Emerging Infectious Diseases and Public Health Security, the Xinjiang Production and Construction Corps, Shihezi 832003, Xinjiang, China.
Biomed Environ Sci. 2024 Oct 20;37(10):1128-1141. doi: 10.3967/bes2024.111.
Mitochondrial reactive oxygen species (mtROS) could cause damage to pancreatic β-cells, rendering them susceptible to oxidative damage. Hence, investigating the potential of the mitochondria-targeted antioxidant (Mito-TEMPO) to protect pancreatic β-cells from ferroptosis by mitigating lipid peroxidation becomes crucial.
MIN6 cells were cultured with 100 μmol/L sodium palmitate (SP) to simulate diabetes. FerroOrange was utilized for the detection of Fe fluorescence staining, BODIPY581/591C11 for lipid reactive oxygen species, and MitoSox-Red for mtROS. Alterations in mitophagy levels were assessed through the co-localization of lysosomal and mitochondrial fluorescence. Western blotting was employed to quantify protein levels of Acsl4, GPX4, FSP1, FE, PINK1, Parkin, TOMM20, P62, and LC3. Subsequently, interventions were implemented using Mito-TEMPO and Carbonyl cyanide 3-chlorophenylhydrazone (CCCP) to observe changes in ferroptosis and mitophagy within MIN6 cells.
We found that SP induced a dose-dependent increase in Fe and lipid ROS in MIN6 cells while decreasing the expression levels of GPX4 and FSP1 proteins. Through bioinformatics analysis, it has been uncovered that mitophagy assumes a crucial role within the ferroptosis pathway associated with diabetes. Additionally, SP decreased the expression of mitophagy-related proteins PINK1 and Parkin, leading to mtROS overproduction. Conversely, Mito-TEMPO effectively eliminated mtROS while activating the mitophagy pathways involving PINK1 and Parkin, thereby reducing the occurrence of ferroptosis in MIN6 cells. CCCP also demonstrated efficacy in reducing ferroptosis in MIN6 cells.
In summary, Mito-TEMPO proved effective in attenuating mtROS production and initiating mitophagy pathways mediated by PINK1 and Parkin in MIN6 cells. Consequently, this decreased iron overload and lipid peroxidation, ultimately safeguarding the cells from ferroptosis.
线粒体活性氧（mtROS）可对胰腺β细胞造成损伤，使其易受氧化损伤。因此，研究线粒体靶向抗氧化剂（Mito-TEMPO）通过减轻脂质过氧化作用来保护胰腺β细胞免受铁死亡的潜力至关重要。
用100μmol/L棕榈酸钠（SP）培养MIN6细胞以模拟糖尿病。利用FerroOrange检测铁荧光染色，BODIPY581/591C11检测脂质活性氧，MitoSox-Red检测mtROS。通过溶酶体和线粒体荧光的共定位评估线粒体自噬水平的变化。采用蛋白质免疫印迹法量化酰基辅酶A合成酶长链家族成员4（Acsl4）、谷胱甘肽过氧化物酶4（GPX4）、铁死亡抑制蛋白1（FSP1）、铁（FE）、PTEN诱导激酶1（PINK1）、帕金蛋白（Parkin）、线粒体外膜转位酶20（TOMM20）、p62和微管相关蛋白1轻链3（LC3）的蛋白水平。随后，使用Mito-TEMPO和羰基氰3-氯苯腙（CCCP）进行干预，观察MIN6细胞中铁死亡和线粒体自噬的变化。
我们发现SP诱导MIN6细胞中铁和脂质活性氧呈剂量依赖性增加，同时降低GPX4和FSP1蛋白的表达水平。通过生物信息学分析发现，线粒体自噬在与糖尿病相关的铁死亡途径中起关键作用。此外，SP降低了线粒体自噬相关蛋白PINK1和Parkin的表达，导致mtROS过度产生。相反，Mito-TEMPO有效消除了mtROS，同时激活了涉及PINK1和Parkin的线粒体自噬途径，从而减少了MIN6细胞中铁死亡的发生。CCCP在减少MIN6细胞中铁死亡方面也显示出效果。
总之，Mito-TEMPO被证明可有效减少MIN6细胞中mtROS的产生，并启动由PINK1和Parkin介导的线粒体自噬途径。因此，这降低了铁过载和脂质过氧化，最终保护细胞免受铁死亡。
